Calcium ions are implicated in a variety of physiological functions, including enzyme activity, membrane excitability, neurotransmitter release, and synaptic transmission, etc. Calcium antagonists have been known to be effective for the treatment of exertional angina and essential hypertension. Selective and nonselective voltage-dependent calcium channel blockers also have inhibitory action on the acute and tonic pain behaviors resulting from thermal stimulation, subcutaneous formalin injection and nerve injury. This study was undertaken to investigate the effects of iontophoretically applied Ca++ and its antagonists on the responses of WDR (wide dynamic range) cells to sensory inputs. The responses of WDR cells to graded electrical stimulation of the afferent nerve and also to thermal stimulation of the receptive field were recorded before and after iontophoretical application of Ca++, EGTA, Mn++, verapamil, omega-conotoxin GVIA, omega-conotoxin MVIIC and omega-agatoxin IVA. Also studied were the effects of a few calcium antagonists on the C-fiber responses of WDR cells sensitized by subcutaneous injection of mustard oil (10%). Calcium ions and calcium channel antagonists (Mn++, verapamil, omega-conotoxin GVIA & omega-agatoxin IVA) current-dependently suppressed the C-fiber responses of WDR cells without any significant effects on the A-fiber responses. But omega-conotoxin MVIIC did not have any inhibitory actions on the responses of WDR cell to A-fiber, C-fiber and thermal stimulation. Iontophoretically applied EGTA augmented the WDR cell responses to C-fiber and thermal stimulations while spinal application of EGTA for about 20 ~ 30 min strongly inhibited the C-fiber responses. The augmenting and the inhibitory actions of EGTA were blocked by calcium ions. The WDR cell responses to thermal stimulation of the receptive field were reduced by imtophoretical application of Ca++, verapamil, omega -agatoxin IVA, and omega-conotoxin GVIA but not by omega-conotoxin MVIIC. The responses of WDR cells to C-fiber stimulation were augmented after subcutaneous injection of mustard oil (10%, 0.15 ml) into the receptive field and these sensitized C-fiber responses were strongly suppressed by iontophoretically applied Ca++, verapamil, omega-conotoxin GVIA and omega-agatoxin IVA. These experimental findings suggest that in the rat spinal cord, L-, N-, and P-type, but not Q-type, voltage-sensitive calcium channels are implicated in the calcium antagonist-induced inhibition of the normal and the sensitized responses of WDR cells to C-fiber and thermal stimulation, and that the suppressive effect of calcium and augmenting action of EGTA on WDR cell responses are due to changes in excitability of the cell.
Animals ; Calcium Channel Blockers* ; Calcium Channels* ; Calcium* ; Egtazic Acid ; Electric Stimulation ; Formaldehyde ; Hypertension ; Injections, Subcutaneous ; Ions ; Iontophoresis ; Membranes ; Mustard Plant ; Neurotransmitter Agents ; omega-Agatoxin IVA ; omega-Conotoxin GVIA ; omega-Conotoxins ; Posterior Horn Cells* ; Rats* ; Spinal Cord ; Synaptic Transmission ; Verapamil

The aim of this study was to investigate the role of Ca2+-channel blockers in norepinephrine (NE) release from rat hippocampus. Slices and synaptosomes were incubated with [3H]-NE and the releases of the labelled products were evoked by 25 mM KCl stimulation. Nifedipine, diltiazem, nicardipine, flunarizine and pimozide did not affect the evoked and basal release of NE in the slice. But, diltiazem, nicardipine and flunarizine decreased the evoked NE release with a dose-related manner without any change of the basal release from synaptosomes. Also, a large dose of pimozide produced modest decrement of NE release. omega-conotoxin (CTx) GVIA decreased the evoked NE release in a dose-dependent manner without changing the basal release. And omega-CTxMVIIC decreased the evoked NE release in the synaoptosomes without any effect in the slice, but the effect of decrement was far less than that of omega-CTxGVIA. In interaction experiments with omega-CTxGVIA, omega-CTxMVIIC slightly potentiated the effect of omega-CTxGVIA on NE release in the slice and synaptosomal preparations. These results suggest that the NE release in the rat hippocampus is mediated mainly by N-type Ca2+-channels, and that other types such as L-, T- and/or P/Q-type Ca2+-channels could also be participate in this process.
Animals ; Diltiazem ; Flunarizine ; Hippocampus ; Nicardipine ; Nifedipine ; Norepinephrine* ; omega-Conotoxins ; Pimozide ; Rats* ; Synaptosomes*

The present study was designed to establish comparatively the inhibitory effects of cilnidipine (CNP), nifedipine (NIF), and omega-conotoxin GVIA (CTX) on the release of CA evoked by cholinergic stimulation and membrane depolarization from the isolated perfused model of the rat adrenal medulla. CNP (3 micrometer), NIF (3 micrometer), and CTX (3 micrometer) perfused into an adrenal vein for 60 min produced greatly inhibition in CA secretory responses evoked by ACh (5.32 x 10(-3) M), DMPP (10(-4) M for 2 min), McN-A-343 (10(-4) M for 2 min), high K+ (5.6 x 10(-2) M), Bay-K-8644 (10(-5) M), and cyclopiazonic acid (10(-5) M), respectively. For the CA release evoked by ACh and Bay-K-8644, the following rank order of potency was obtained: CNP > NIF > CTX. The rank order for the CA release evoked by McN-A-343 and cyclopiazonic acid was CNP > NIF > CTX. Also, the rank orders for high K+ and for DMPP were NIF > CTX > CNP and NIF > CNP > CTX, respectively. Taken together, these results demonstrate that all voltage-dependent Ca2+ channels (VDCCs) blockers of cilnidipine, nifedipine, and omega-conotoxin GVIA inhibit greatly the CA release evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors and the membrane depolarization without affecting the basal release from the isolated perfused rat adrenal gland. It seems likely that the inhibitory effects of cilnidipine, nifedipine, and omega-conotoxin GVIA are mediated by the blockade of both L- and N-type, L-type only, and N-type only VDCCs located on the rat adrenomedullary chromaffin cells, respectively, which are relevant to Ca2+ mobilization. It is also suggested that N-type VDCCs play an important role in the rat adrenomedullary CA secretion, in addition to L-type VDCCs.
(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride ; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; Adrenal Glands ; Adrenal Medulla* ; Animals ; Calcium Channels ; Calcium Channels, L-Type ; Calcium Channels, N-Type ; Chromaffin Cells ; Dimethylphenylpiperazinium Iodide ; Membranes ; Nifedipine* ; omega-Conotoxin GVIA* ; omega-Conotoxins* ; Rats* ; Veins

Animals ; Calcium ; metabolism ; Cell Hypoxia ; Cells, Cultured ; Hippocampus ; cytology ; Neurons ; drug effects ; metabolism ; Rats ; Rats, Wistar ; omega-Conotoxins ; pharmacology

Flavonoids have an ability to suppress various ion channels. We determined whether one of flavonoids, cyanidin-3-glucoside, affects adenosine 5'-triphosphate (ATP)-induced calcium signaling using digital imaging methods for intracellular free Ca2+ concentration ([Ca2+]i), reactive oxygen species (ROS) and mitochondrial membrane potential in PC12 cells. Treatment with ATP (100microM) for 90 sec induced [Ca2+]i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside (1micro g/ml to 100microg/ml) for 30 min inhibited the ATP-induced [Ca2+]i increases in a concentration-dependent manner (IC50=15.3microg/ml). Pretreatment with cyanidin-3-glucoside (15microg/ml) for 30 min significantly inhibited the ATP-induced [Ca2+]i responses following removal of extracellular Ca2+ or depletion of intracellular [Ca2+]i stores. Cyanidin-3-glucoside also significantly inhibited the relatively specific P2X2 receptor agonist 2-MeSATP-induced [Ca2+]i responses. Cyanidin-3-glucoside significantly inhibited the thapsigargin or ATP-induced store-operated calcium entry. Cyanidin-3-glucoside significantly inhibited the ATP-induced [Ca2+]i responses in the presence of nimodipine and omega-conotoxin. Cyanidin-3-glucoside also significantly inhibited KCl (50 mM)-induced [Ca2+]i increases. Cyanidin-3-glucoside significantly inhibited ATP-induced mitochondrial depolarization. The intracellular Ca2+ chelator BAPTA-AM or the mitochondrial Ca2+ uniporter inhibitor RU360 blocked the ATP-induced mitochondrial depolarization in the presence of cyanidin-3-glucoside. Cyanidin-3-glucoside blocked ATP-induced formation of ROS. BAPTA-AM further decreased the formation of ROS in the presence of cyanidin-3-glucoside. All these results suggest that cyanidin-3-glucoside inhibits ATP-induced calcium signaling in PC12 cells by inhibiting multiple pathways which are the influx of extracellular Ca2+ through the nimodipine and omega-conotoxin-sensitive and -insensitive pathways and the release of Ca2+ from intracellular stores. In addition, cyanidin-3-glucoside inhibits ATP-induced formation of ROS by inhibiting Ca2+-induced mitochondrial depolarization.
Adenosine ; Adenosine Triphosphate ; Animals ; Calcium ; Calcium Signaling ; Flavonoids ; Ion Channels ; Ion Transport ; Membrane Potential, Mitochondrial ; Nimodipine ; omega-Conotoxins ; PC12 Cells* ; Reactive Oxygen Species ; Receptors, Purinergic P2X2 ; Thapsigargin

Excitatory amino acid (EAA) and substance P (SP) have been known to be primary candidates for nociceptive neurotransmitter in the spinal cord, and calcium ions are implicated in processing of the sensory informations mediated by EAA and SP in the spinal cord. In this study, we examined how Ca2+ modified the responses of dorsal horn neurons to single or combined iontophoretical application of EAA and SP in the rat. All the LT cells tested responded to kainate, whereas about 55% of low threshold (LT) cells responded to iontophoretically applied NMDA. NMDA and kainate excited almost all wide dynamic range (WDR) cells. These NMDA- and kainate-induced WDR cell responses were augmented by iontophoretically applied EGTA, but suppressed by Ca2+, Mn2+ verapamil and omega-conotoxin GVTA, effect of verapamil being more prominent and well sustained. Ca2+ and Mn2+ antagonized the augmenting effect of EGTA. On the other hand, prolonged spinal application of EGTA suppressed the response of WDR cell to NMDA. SP had triple effects on the spontaneous activity as well as NMDA-induced responses of WDR cells: excitation, inhibition and no change. EGTA augmented, but Ca2+, Mn2+ and verapamil suppressed the increase in the NMDA-induced responses and spontaneous activities of WDR cells following iontophoretical application of SP. These results suggest that in the spinal cord, sensory informations mediated by single or combined action of EAA and SP can be modified by the change in calcium ion concentration.
Animals ; Calcium* ; Egtazic Acid ; Excitatory Amino Acids* ; Hand ; Ions ; Iontophoresis ; Kainic Acid ; N-Methylaspartate ; Neurotransmitter Agents ; omega-Conotoxins ; Posterior Horn Cells* ; Rats* ; Spinal Cord ; Substance P ; Verapamil

Amino Acid Sequence ; Animals ; Calcium Channel Blockers ; pharmacology ; Calcium Channels ; drug effects ; Humans ; Molecular Sequence Data ; Structure-Activity Relationship ; omega-Conotoxins ; chemistry ; pharmacology

The Conus venom is secreted by the duct and theca of venom. Most of conotoxins are composed of 10-40 amino acid residues with several disulfide bridges. They can specifically target neurotransmitter receptors including nAChRs, calcium ion channels, sodium ion channels and potassium ion channels, etc. Some conotoxins, such as that target N-Ca2+ channels, nAChR alpha9alpha10 subtype, TTX-R Na+ channels or NMDA receptors, have potent antinociceptive activities, omega-MVIIA, an Ca2+ channels blocker was approved by FDA in December, 2004 for marketing. Because of lower molecular weight and high specificity, conotoxins are the powerful pharmacology tools and potent analgesics without addiction. This review briefly summarizes the research progress of antinociceptive conotoxins and addresses on their targets and structure-activity relationships.
Analgesics ; pharmacology ; Calcium Channels ; drug effects ; Conotoxins ; pharmacology ; Sodium Channels ; drug effects ; Structure-Activity Relationship

The α3* nAChRs, which are considered to be promising drug targets for problems such as pain, addiction, cardiovascular function, cognitive disorders etc., are found throughout the central and peripheral nervous system. The α-conotoxin (α-CTx) LvIA has been identified as the most selective inhibitor of α3β2 nAChRs known to date, and it can distinguish the α3β2 nAChR subtype from the α6/α3β2β3 and α3β4 nAChR subtypes. However, the mechanism of its selectivity towards α3β2, α6/α3β2β3, and α3β4 nAChRs remains elusive. Here we report the co-crystal structure of LvIA in complex with Aplysia californica acetylcholine binding protein (Ac-AChBP) at a resolution of 3.4 Å. Based on the structure of this complex, together with homology modeling based on other nAChR subtypes and binding affinity assays, we conclude that Asp-11 of LvIA plays an important role in the selectivity of LvIA towards α3β2 and α3/α6β2β3 nAChRs by making a salt bridge with Lys-155 of the rat α3 subunit. Asn-9 lies within a hydrophobic pocket that is formed by Met-36, Thr-59, and Phe-119 of the rat β2 subunit in the α3β2 nAChR model, revealing the reason for its more potent selectivity towards the α3β2 nAChR subtype. These results provide molecular insights that can be used to design ligands that selectively target α3β2 nAChRs, with significant implications for the design of new therapeutic α-CTxs.
Animals ; Aplysia ; Binding Sites ; Conotoxins ; chemistry ; Crystallography, X-Ray ; Humans ; Protein Structure, Quaternary ; Receptors, Nicotinic ; chemistry

Glutamate Is the predominant excitatory neurotransmitter in the mammalian CNS. To elucidate the influence of glutamate on the noradrenergic neurotransmission in rat cortex, we examined the effects of agents that act in several steps of neurotransmission on [3H]norepinephrine ([3H])NE) release evoked by glutamate. Glutamate (1 mM) evoked significant release of [3H]NE from rat cortex slices in the absence of Mg2+in the incubation media. This effect was attenuated by cromakalime (10 nM) and lemakalime (10 nM), and the inhibitory effect of cromakalime was abolished by glipizide. Inhibitory effect of muscimol (30 uM) and baclofen (3 uM, 30 uM) was antagonized by biccuculine (3 uM), respectively. Nipecotic acid(10 uM), DABA(300 uM), and beta-alanine(100 uM) attenuated the glutamate-induced release of [3H]NE. Dihydrokinate (300 uM) PDC (100 nM) increased the glutamate-induced release of [3H]NE. Ifenprodile (10 nM) and arcaine (1 uN), blockers of polyamine site, attenuated the release of ("H)NE. The stimulatory effect of spermine was abolished by arcaine. CPA(100 nM) and CPCA(100 nM), EHNA(30 uN) and NBTI(1 uN) attenuated the release of ("H)NE. Verapamil(S uN), nitredipine(10 uN), u- conotoxin (100 nM) and flunarizine (5 uM) attenuated the release of (3H)NE. Dantrolene(30 uM), KT-362(3 uM), and ryanodine(10 nM), attenuated the glutamate-induced release of [3H]NE. Glycine (10 uM) increased the release of [3H]NE. DCQX (30 uN) attenuated the release of [3H]NE. These results suggest that glutamate-evoked release of norepinephrine can be modulated by GABAergic, adenosinergic neurotransmitters, and by various drugs which modulate ion channel activities in rat cortex.
Animals ; Baclofen ; Cerebral Cortex ; Conotoxins ; Cromakalim ; Flunarizine ; Glipizide ; Glutamic Acid ; Glycine ; Ion Channels ; Muscimol ; Neurotransmitter Agents ; Norepinephrine* ; Rats* ; Spermine ; Synaptic Transmission